Little is known about the role of microbial biofilms in nutrient cycling and ecosystem processes within desert springs. However, biofilms produce microscale physicochemical variation important to ecosystem function. We used microelectrodes to measure microscale physicochemical (temperature, pH, O₂, and H₂S) heterogeneity in biofilms at Devils Hole, Nevada. Additionally, we measured water column and pore water nutrient concentrations in 2 autotrophic (Spirogyra, cyanobacteria) and 1 heterotrophic (Beggiatoa) biofilm types. Spirogyra and cyanobacteria followed similar physicochemical trends; however, Spirogyra had more pronounced diurnal and seasonal variation. Oxygen concentrations within the biofilms varied with sample month, light availability, and biofilm type. We also assessed the nutrient limitation of desert spring microbial biofilms using a nutrient-diffusing substrata experiment. Autotrophic and heterotrophic biofilms responded differently to nutrient treatments. The differences between cyanobacteria and Spirogyra have implications on the ecosystem function of desert springs, as well as other ecosystems with diverse biofilm communities, and suggest that a change in microbial biofilms could alter ecosystem biogeochemical dynamics.